[2153] Effect of Decalcification Agents on Nucleic Acid Quantity and Quality
Veena M Singh, Ranelle C Salunga, Yen K Tran, Jose Gallindo, Pamela A Plumlee, Sunny W Chu, Mark G Erlander, Michael R Peterson. University of California San Diego, San Diego; bioTheranostics, San Diego
Background: Molecular studies are part of standard care for cancer patients. Bone a common or sole site of metastasis requires decalcification. However commonly used decalcification agents usually contain strong acids that degrade RNA/DNA. We propose to systematically study the effect of a spectrum of commercial decalcifiers (strong acid-hydrochloric/nitric, weak acid-formic and chelating agent-EDTA) on the quantity/quality of RNA/DNA. The paradigm shift in oncology with biomarker targeted therapy and gene expression profiling studies, necessitates improved nucleic acid quality/quantity from bone.
Design: Multiple bone biopsies (obtained from amputations) of similar size and cellularity were fixed in 10% neutral buffered formalin, randomized to various decalcifiers for 2 hours then processed and embedded. Tissue lysates were obtained from unstained sections and nucleic acid isolated. DNA/RNA was quantified in nanograms by PicoGreen and RiboGreen (Life Technologies,Carlsbad,CA) respectively. Assessment of DNA/RNA integrity was accomplished by comparison of the average cycle threshold (CT) by PCR of selected housekeeping genes for each decalcifier. Results were then analyzed by two sample t-test.
Results: There was a significant decrease in both DNA/RNA yield and integrity with strong acids (hydrochloric, nitric) vs. 14% EDTA and formic acid. DNA yield was (mean ng) 5.9 vs. 37.1 (p-value 0.014) and RNA was (mean ng) 39.5 vs. 157.9 (p-value 0.002). DNA integrity was (mean CT) 38.7 vs. 28.9 (p-value 0.00004) and RNA was 34.9 vs. 26.3 (p-value 0.002). A 1-2 CT improvement for 14% EDTA and immunocal was observed.
Conclusions: The decalcification agents associated with the greatest nucleic acid quantity and better quality are 14% EDTA and formic acid decalcifiers. In addition data suggests that RNA integrity improves with 14% EDTA or immunocal.
| DNA Quantity(Picogreen) | DNA Quality (qPCR)Equal ng/reaction | RNA Quantity (Ribogreen) | RNA Quality (qRT-PCR) Equal ng/reaction | ||
| Commercial Name | Decalcification agent | Yield (ng) | Ave CT | Yield (ng) | Ave CT |
| 14% EDTA | 14% EDTA | 67.8 | 28.3 | 226.2 | 25.19 |
| Immunocal | Formic acid | 41.5 | 29.06 | 175.0 | 25.24 |
| Surgpath decalcifier I | Formaldehyde/Methanol/Formic acid | 27.1 | 29.53 | 144.8 | 26.43 |
| Formical4 | Formic acid/Formaldehyde/EDTA | 26.4 | 29.2 | 142.6 | 27.31 |
| Formical2000 | Formic acid/EDTA | 22.8 | 28.16 | 101.0 | 27.44 |
| Nitrical | Nitric acid | 11.4 | 39.6 | 51.7 | 31.3 |
| IMEB HCL/Formic decal solution | HCL/Formic acid | 5.8 | 36.37 | 50.4 | 38.8 |
| Surgpath decalcifier II | HCL/EDTA | 5.3 | 40.0 | 30.5 | 34.8 |
| Decal stat | HCL/EDTA | 1.4 | Quantity Not Sufficient | 25.4 | Quantity Not Sufficient |